Abstract
The establishment of traits that result from the concerted expression of complementing transgene fragments is a feasible tool for trait control or gene flow control in plants. This chapter describes the methodology for producing herbicide-resistant and pollen-sterile wheat plants by the intein-mediated assembly of inactive precursor protein fragments (protein trans-splicing). We suggest the design of intein-containing vectors for split-transgene expression. We describe transient plant assays that can be used to analyse the functionality of the system and describe the transformation of wheat plants using a split selection marker.
We hope that this chapter will be a helpful guideline for researchers who are interested in applying similar split-gene approaches in wheat or other monocotyledonous crops.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Evans, T.C., Jr., Xu, M. Q., and Pradhan, S. (2005) Protein splicing elements and plants: from transgene containment to protein purification. Annu. Rev. Plant Biol. 56, 375–392.
Gleba, Y., Marillonnet, S., and Klimyuk, V. (2004) Design of safe and biologically contained transgenic plants: tools and technologies for controlled transgene flow and expression. Biotechnol. Genet. Eng. Rev. 21, 325–567.
Burgess, D. G., Ralston, E. J., Hanson, W. G., Heckert, M., Ho, M., Jenq, T., Palys, J. M., Tang, K., and Gutterson, N. (2002) A novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants. Plant J. 31, 113–125.
Perler, F.B. (1998) Protein splicing of inteins and hedgehog autoproteolysis: structure, function, and evolution. Cell 92, 1–4.
Chin, H.G., Kim, G-D., Marin, I., Mersha, F., Evans, Jr., T. C. Chen, L., Xu, M-Q., and Pradhan, S. (2003) Protein trans-splicing in transgenic plant chloroplast: reconstruction of herbicide resistance from split genes. Proc. Natl. Acad. Sci. USA 100, 4510–4515.
Dun, B. Q., Wang, X. J., Lu, W., Zhao, Z. L., Hou, S. N., Zhang, B. M., Li, G. Y., Evans, T. C. Jr, Xu, M. Q., and Lin, M. (2007) Reconstitution of glyphosate resistance from a split 5-enolpyruvyl shikimate-3-phosphate synthase gene in Escherichia coli and transgenic tobacco. Appl. Environ. Microbiol. 73, 7997–8000.
Gils, M., Marillonnet, S., Werner, S., Grützner, R., Giritch, A., Engler, C., Schachschneider, R., Klimyuk, V., and Gleba, Y.. (2008) A novel hybrid seed system for plants. Plant Biotechnol. J. 6, 226–235.
Yang, J., Fox, Jr., G. C., and Henry-Smith, T. V. (2003) Intein-mediated assembly of a functional b-glucuronidase in transgenic plants. Proc. Natl. Acad. Sci. USA 100, 3513–3518.
Kempe, K., Rubtsova, M., and Gils, M. (2009) Intein-mediated protein assembly in transgenic wheat: production of active barnase and acetolactate synthase from split genes. Plant Biotechnol. J. 7, 283–97.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15, 473–497.
Xu, M.Q. and Perler, F. B. (1996) The mechanism of protein splicing and its modulation by mutation. EMBO J. 15, 5146–5153.
Evans, T.C., Jr., Martin, D., Kolly, R., Panne, D., Sun, L., Ghosh, I., Chen, L., Benner, J., Liu, X. Q., and Xu, M. Q. (2000) Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synecho-cystis species PCC6803. J. Biol. Chem. 275, 9091–9094.
Alexander, M.P., (1969) Differential staining of aborted and nonaborted pollen. Stain Technol. 44, 117–122.
Koncz, C. and Schell, J. (1986) The promoter of the TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383–396.
Acknowledgements
The authors sincerely wish to thank Angelika Gils for her critical reading of the manuscript.
The research was funded by the Bundesministerium für Bildung und Forschung (BMBF, GABI-FUTURE grant 0315043A) at the Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Gatersleben, and in cooperation with the Nordsaat GmbH, Böhnshausen, Germany.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Gils, M., Rubtsova, M., Kempe, K. (2012). Split-Transgene Expression in Wheat. In: Dunwell, J., Wetten, A. (eds) Transgenic Plants. Methods in Molecular Biology, vol 847. Humana Press. https://doi.org/10.1007/978-1-61779-558-9_11
Download citation
DOI: https://doi.org/10.1007/978-1-61779-558-9_11
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-557-2
Online ISBN: 978-1-61779-558-9
eBook Packages: Springer Protocols